The present application is a reissue of Ser. No. 09/061,036, filed Apr. 16, 1998, now U.S. Pat. No. 6,149,890.
The present invention relates to the field of the diagnostic technique known as Magnetic Resonance Imaging (M.R.I.), a renowned, powerful diagnostic procedure used in medical practice to detect anomalies in organs or tissues of the human or animal body (see: Rocklage S. M., Watson A. D. and Carvlin M. J., Magnetic Resonance Imaging, Chap. 14, Vol. 1, Second Ed., 1992; Stark D. D. and Bradley W. G. Eds.). Particularly, the invention relates to novel compounds able to chelate paramagnetic metal ions, to their chelated complexes and to the physiologically acceptable salts thereof as well as the use of these compounds as M.R.I. contrast agents.
Lanthanide ions are known to be among the most toxic paramagnetic metal ions in vivo. It is therefore necessary to administer said ions in the form of chelated complexes to the subject undergoing such as a diagnostic procedure, as chelation by an electron donor ligand appreciably decreases the toxicity of the metal ion. On the other hand, this often also decreases undesirably the relaxivity properties, as accessibility by the water protons to the inner coordination sphere of the metal ion is reduced. The relaxivity of a chelated complex is generally significantly lower than that of the corresponding metal ion (The British Journal of Radiology, 68, 225-247, 1995).
It is therefore important for the paramagnetic chelated complex to have a high stability, i.e. a high thermodynamic stability constant, in order to have a low toxicity. A higher stability can be obtained, for example, using macrocyclic ligands, in which the metal ion is incorporated in a rigid structure; Gd-DOTA (Dotarem(copyright)) or Gd-HP-DO3A (ProHance(copyright)) are, for example, known to have a higher stability than Gd-DTPA (Investigative Radiology, 27 (Suppl.1), S1-S6, 1992; Topics in Magnetic Resonance Imaging, 7(3), 181-195, 1995). Gd-DOTA and GD-HP-DO3A are macrocyclic ligand contrast agents commercially available at present.
The thermodynamic stability being the same, other factors affecting the toxicity of a contrast agent are the osmolality of the solution (in case of injectable formulations) and the intrinsic toxicity of the molecule, or molecular toxicity (Toxicology Letters, 64/65, 705-715, 1992). The osmolality of the solution particularly affects the toxicity of the contrast agent after intravenous administration; the molecular toxicity should, in its turn, be specifically considered when the contrast agent is administered to patients with impaired permeability of the blood-brain barrier, for example patients affected by cerebrovascular disorders, cerebral metastases, traumas and the like, in that the contrast agent can enter the tissues of central nervous system (Investigative Radiology, 25, S49-S50, 1990; M. Nadjmi Ed., XVth Congress of the European Society of Neuroradiology, Wxc3xcrzburg, Sept. 13-17th, 1988, 581-584 xe2x80x94Springer-Verlag Berlin Heidelberg 1989); this can, in fact, result in the appearance of even remarkable neurotoxic effects. Molecular toxicity can be evaluated by tests carried out either in vitro (histamine release, inhibition of enzymatic activity and of coagulation); or in vivo, administering the compound directly to the nervous tissue, which is the substrate most sensitive to molecular toxicity.
DL50 values after intracerebral administration (intracisternal, intracerebroventricular), for example in the mouse and in the rat, can therefore be taken as a highly sensitive index of the molecular toxicity of the contrast agent (Toxicology Letters, cited ref.). In particular, the intracerebroventricular administration seems to be the most sensitive to discriminate the neurotoxicity of the compounds (Proceedings of the 10th National Congress of the Italian Toxicology Society, Pavia, September 21-24th, 100, 1994).
The xe2x80x9cneurotoxicity indexxe2x80x9d is defined by the following ratio:       neurotoxicity    ⁢          xe2x80x83        ⁢    index    =                    DL        50            ⁢              i        .        v                            DL        50            ⁢              i        .        c        .            
It is evident that, DL50s values after intravenous administration being the same, the higher the DL50 value after intracerebral administration, the lower the neurotoxicity index of the compound.
Generally speaking, open-chain paramagnetic chelates have a cerebral toxicity different than the corresponding macrocyclic chelates; in particular, chelates such as Magnevist(copyright) (gadopentetate dimeglumine) and Omniscan(copyright) (gadodiamide) showed a better tolerability, after intracisternal administration, than such macrocyclic chelates as Dotarem(copyright) (gadoterate), gadobutrol and ProHance(copyright) (gadoteridol) (European Journal of Radiology, 21, 1-10, 1995).
Researches in the field of M.R.I. contrast agents are therefore directed to discovery of chelating agents having a high stability to paramagnetic metal ions, with a consequent decrease in toxicity deriving from the release of the free metal ion, which keep a good relaxivity and a low effective dose and which, above all in case of specific uses, have a low neurotoxicity index. This proves of paramount importance when the administration of high doses of contrast agent is necessary in order to improve the imaging of some lesions, such as infections, metastases or cerebral neoplasms, sub-acute cerebral infarction, head and neck tumors (Topics in Magnetic Resonance Imaging, 7(3), 181-195, 1995; The British Journal of Radiology, 68, 225-247, 1995).
The present invention relates to chelated complexes of paramagnetic metals characterized by an extremely favourable neurotoxicity profile. In particular, the paramagnetic chelated complexes of the present invention showed, after intracerebroventricular administration to the mouse, extremely high DL50 values compared with the teachings of the prior art. The compound described in example 1 (gadolinium complex of 10-[2-[[2-(2-hydroxyethoxy)ethyl]amino]-2-oxoethyl]-xcex1,xcex1xe2x80x2,xcex1xe2x80x3-tris(hydroxymethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic) acid has, for example, a DL50 of 0.23 mmol/kg after intracerebroventricular administration to the mouse. The compounds described in example 2 (gadolinium complex of xcex1,xcex1xe2x80x2,xcex1xe2x80x3-tris (hydroxymethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) and in Example 4 (Na+Gd[THM-DOTA]xe2x88x92) have a very good tolerability as well, with a DL50, after intracerebroventricular administration to the mouse, of 0.170 and 0.150 mmol/kg, respectively; the corresponding value for Gd-DOTA being 0.064 mmol/kg. This strongly suggests the possible use of the compounds of the present invention for imaging of cerebral lesions such as tumors, metastases, in highly safe conditions for the patient.
The present invention relates to the compounds of formula (I), both in the racemic and optically active forms: 
wherein:
R is a (C1-C15) straight or branched alkyl chain, optionally interrupted by one or more oxygen, nitrogen, sulfur atoms, as well as by xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NHxe2x80x94 groups, or optionally substituted by one or more NH2, OH, halogen, COOH groups and corresponding ester or amide derivatives; said chain being optionally interrupted and/or substituted by one or more 5- or 6-membered cyclic, saturated, carbocyclic or heterocyclic groups, in which said cyclic groups are optionally substituted by one or more X groups, which can be the same or different, in which
X is xe2x80x94OH, halogen, xe2x80x94NH2, xe2x80x94NHR5, xe2x80x94N(R5)2, xe2x80x94Oxe2x80x94R5, xe2x80x94Sxe2x80x94R5, xe2x80x94COxe2x80x94R5, wherein R5, which can be the same or different, are a (C1-C5) straight or branched alkyl, optionally substituted by one or more hydroxy, alkoxy, carboxy groups, or X is a COOH group, or an ester or amide derivative thereof, or a xe2x80x94SO3H group or an amide derivative thereof,
R1, which can be the same or different, are a hydrogen atom or a xe2x80x94CH2OH group,
with the provisos that:
R is different from: unsubstituted alkyl, xe2x80x94CH2COOH, 
at least two of the R1 substituents are xe2x80x94CH2OH.
The invention also relates to the compounds of formula (II), both in the racemic and optically active forms: 
wherein:
R2 is a hydrogen atom, or a (C1-C15) straight or branched alkyl chain, saturated or unsaturated, optionally interrupted by one or more oxygen, nitrogen, sulfur atoms, as well as by xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2, xe2x80x94SO2NHxe2x80x94 groups, or optionally substituted by one or more NH2, OH, halogen, COOH groups and corresponding ester or amide derivatives; said chain being optionally interrupted and/or substituted by one or more 5- or 6-membered cyclic, carbocyclic or heterocyclic groups, in which said cyclic groups are optionally substituted by one or more X groups, which can be the same or different, in which
X is xe2x80x94OH, halogen, xe2x80x94NH2, xe2x80x94NHR5, xe2x80x94N(R5)2, xe2x80x94Oxe2x80x94R5, xe2x80x94Sxe2x80x94R5, xe2x80x94COxe2x80x94R5, wherein R5, which can be the same or different, are a (C1-C5) straight or branched alkyl, optionally substituted by one or more hydroxy, alkoxy, carboxy groups, or X is a COOH group, or an ester or amide derivative thereof, or a group xe2x80x94SO3H or an amide derivative thereof,
R3, which can be the same or different, are a hydrogen atom or a xe2x80x94CH2OH group,
R4, which can be the same or different, have the same meanings as described for R3 or are CH3 or C2H5,
with the proviso that at least two R3 are xe2x80x94CH2OH.
Within formula (II), a class of particularly preferred compounds comprises those of formula (III): 
wherein:
Y is a xe2x80x94OH group or a xe2x80x94N(R6)2 group, in which R6 groups, which can be the same or different, are a hydrogen atom or a (C1-C15) straight or branched alkyl chain, optionally interrupted by one or more oxygen, nitrogen atoms, as well as by xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NHxe2x80x94 groups, or optionally substituted by one or more NH2, OH, COOH groups and corresponding ester or amide derivatives, or the two R6 groups, taken together, form a cyclic unit, comprising the amide nitrogen atom, being said cyclic unit optionally interrupted by one or more oxygen and/or nitrogen atoms and optionally substituted by one or more X groups, which can be the same or different, wherein X has the same meanings as described for the compounds of formula (II);
R3, which can be the same or different, are a hydrogen atom or a xe2x80x94CH2OH group;
with the proviso that at least two R3 are xe2x80x94CH2OH.
Objects of the invention also are:
the optically active forms of the compounds of formulae (I), (II) and (III), when chiral centres are present;
chelated complexes of the compounds of formulae (I), (II) and (III) with the ions of metallic elements having atomic numbers ranging from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83, among which particularly preferred are: Gd(III), Mn(II), Fe(II), Fe(III), Cu(II), Cr(III), Eu(III), Dy(III), La(III), Yb(III);
the salts thereof with physiologically acceptable organic bases selected from primary, secondary, tertiary amines or basic amino acids, or with inorganic bases the cations of which are sodium, potassium, magnesium, calcium, or mixtures thereof, or with anions of physiologically acceptable organic acids, or with anions of inorganic acids such as hydrogen halides.
It is also an object of the invention the use of the compounds of formula (I), (II) and (III) and of the complex salts thereof for the preparation of pharmaceutical compositions for the diagnostic use, as well as the formulations themselves.
In compounds of formula (I), particularly preferred meanings for R are the following: 
In compounds of formula (II), particularly preferred meanings for R2 are the following: 
In compound of formula (III), particularly preferred meanings for Y are the following: 
The compounds of the invention are valuable for use in a number of different fields. Non limiting examples of the uses thereof are: recovery, separation, selective extraction of metal ions; use in therapy as detoxifying or radiotherapy agents; use as contrast agents for the in vivo or in vitro diagnosis through magnetic resonance, X-rays, ultrasounds or scintigraphy.
As M.R.I. contrast agents, preferably used are the chelated complexes of the chelating agents of general formulae (I) to (III) with divalent or trivalent ions of elements having atomic numbers ranging from 21 to 29, 39, 42, 44, or from 57 to 71; Fe(2+), Fe(3+), Cu(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+) or Mn(2+) being preferred; Gd(3+), Mn(2+), Dy(3+) and Fe(3+) being particularly preferred.
For use in X-ray or ultrasound imaging, the chelated metal species is preferably a heavy metal, for example a non-radioactive metal with atomic number higher than 37.
For use in scintigraphy and radiotherapy, the chelated metal species is a radioisotope, such as 51Cr, 68Ga, 111In, 99mTc, 140La, 168Yb.
For use in the detoxication from heavy metals, the ligands of the invention can be administered in the form of salts with physiologically acceptable ions, such as Na+, Ca++, NH4+, Zn++, or as meglumine salts.
For the uses mentioned above, the compounds of the invention can be used as such, or they can be conjugated with macromolecules or incorporated in structures which carry them to specific body sites.
In case the chelated complex has a total charge, this is preferably neutralized with a physiologically acceptable counter-ion. Among the substances suitable for salifying the compounds of the invention and/or their chelated complexes, the following can be cited:
anions of physiologically acceptable inorganic acids, such as hydrogen halides (chlorides, bromides, iodides) or other ions such as sulfate;
anions of organic acids commonly used in the pharmaceutical technique for the salification of basic substances, such as acetate, succinate, citrate, fumarate, maleate, oxalate;
cations of inorganic bases such as ions of alkali or alkaline-earth metals selected from sodium, potassium, magnesium, calcium, and/or mixtures thereof;
cations of physiologically acceptable organic bases selected from primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine;
cations and anions of amino acids such as lysine, arginine and ornithine, or of aspartic and glutamic acids.
Particularly preferred are N-methylglucamine salts.
As far as the administration route is concerned, the compounds of the present invention can be administered by the intravasal (for example intravenous, intraarterial, intracoronaric, intraventricular etc.), intrathecal, intraperitoneal, intralymphatic, intracavital and intraparenchymal routes. They are also suitable for the oral or enteral administration (specifically for the imaging of the gastrointestinal tract) or for the direct injection into a body cavity having a canal communicating with the outside (for example uterus, bladder).
Particularly preferred are anyway the intravasal and, above all, the intrathecal routes, thanks to the unique characteristics of tolerability of the compounds of the invention.
They can be formulated with additives conventionally used for the pharmaceutical or veterinary formulations, for example stabilizers, antioxidants, osmolality and pH adjusters, buffers and the like.
For the parenteral administration, they are preferably formulated as sterile aqueous solutions or suspensions, whose pH can range from 6.0 to 8.5. Said aqueous solutions or suspensions can be administered in concentrations ranging from 0.002 to 1.0 M.
These formulations can also be lyophilized and supplied as such, or for reconstitution before use. For the gastrointestinal use or for the injection into body cavities, these agents can be formulated as a solution or suspension containing suitable additives in order to, for example, control viscosity.
For the oral administration they can be formulated according to preparation methods routinely used in the pharmaceutical technique, optionally also as coated formulations to gain extra protection from the acid pH of stomach, thereby inhibiting the release of the chelated metal ion, which usually occurs at the typical pH values of gastric juices.
Other excipients, such as sweeteners and/or flavours, can also be added according to known techniques of pharmaceutical technique.
The solutions or suspensions of the compounds of this invention can also be formulated as aerosol for use in aerosol-bronchography.
The compounds of formula (I) can preferably be prepared according to the following general synthetic scheme: 
wherein:
R1xe2x80x2=H or xe2x80x94CH2xe2x80x94OPg, wherein Pg is a protective group, for example benzyl;
R, R1 are as defined above for the compounds of formula (I);
X=Cl, Br, I;
The starting product is a compound of formula (1), prepared according to what described in WO 89/05802, wherein the hydroxy groups are protected by a suitable group, for example (in the described case) benzyl. In step:
(a1) said product is reacted with compound R-X, wherein R is the group to be substituted at the tetraazacyclododecane nitrogen and X is an appropriate leaving group, for example Cl, Br; the reaction is preferably carried out in H2O or DMF, at temperatures from 20 to 100xc2x0 C., thereby obtaining compound of formula (2) which, in step
(a2) is deprotected by catalytic hydrogenation, which can be carried out in water with Pd/C at room temperature, to give the desired ligand (3), which, in step
(a3) is reacted with the stoichiometric amount of metal, in the form of salt or oxide, optionally in the presence of the amount of acid or base necessary for the neutralization; the reaction being preferably effected in water or in a suitable water-alcohol mixture, at temperatures from 25 to 100xc2x0 C., preferably from 40 to 80xc2x0 C.; thereby obtaining the chelated complex (4), in which:
Men+=ion of the metallic element having atomic number ranging from 20 to 31, 39, from 42 to 44, 49 and from 57 to 83 (for ex. Gd3+);
n=number of the positive charges of said ion;
m=number of the total charges of the chelated complex;
B=substance able to salify the chelated complex (for example Na+, K+, Mg++, Ca++ or mixtures thereof, meglumine, etc.);
z=number of the charges of B;
p is a number so that the product; pxc2x7z=m.
In case the product of formula (I) is a compound in which R is a mono- or polyhydroxyalkyl chain unsubstituted at the carbon atom adjacent to the macrocycle nitrogen, a different synthetic route can be followed, in which, in step
(b1) compound (1) is reacted in basic medium (for ex. KOH) with a suitable epoxide, in which Rxe2x80x2 can be, for example: H, xe2x80x94CH3, xe2x80x94CH2OH, xe2x80x94CH2xe2x80x94CH2OH, xe2x80x94CHOHxe2x80x94CH2OH, xe2x80x94CHOHxe2x80x94CHOHxe2x80x94CH2OH, 
to obtain a compound (5) which, in step
(b2) is deprotected at the hydroxyls according to what described in step (a2); the resulting compound (6) is reacted in step
(b3) with the suitable salt or oxide of the desired metal, according to the general procedure described above, to give the corresponding chelated complex (7).
On the other hand, in case compound of formula (I) is a compound in which R is a mono- or polyhydroxyalkyl chain substituted at the carbon atom adjacent to the macrocycle nitrogen, in step
(c1) compound (1) is reacted in basic medium (for ex. KOH) with a suitable epoxide, in which Rxe2x80x2 and Rxe2x80x3 have independently the meanings described above for Rxe2x80x2, except for H, to obtain compound (8) which, after deprotection and complexation, gives the final compound (10).
Concerning the preparation of the compounds of formulae (II) and (III), the synthetic route differs depending on the positions on the ring at which the hydroxymethyl substituents are to be introduced. The following synthetic routes can be used:
1) Synthesis known as xe2x80x9ccrab-likexe2x80x9d (Tetrahedron Letters, 31, 1077-1080, 1990; Synlett, 611-620, 1993).
The general scheme for the synthesis of the ligand described in example 3 and of the its analogues is reported in the following: 
The starting compound is 2-chloro-3-hydroxy-propionic acid chloride (1), wherein the hydroxyl is protected by a suitable group, preferably benzyl. This compound is reacted with bis(phenylmethyl)ethylene-diamine (2) and NaI, to give the intermediate (3) which, by reaction with bis (phenylmethyl)ethylenediamine, cyclizes, yielding the intermediate (4). This is reduced at the carbonyl groups, to give compound (5) which, upon deprotection of the hydroxyls, gives intermediate (6).
Intermediate (6) is then alkylated with the xcex1-halo derivative of a suitable carboxylic acid, to obtain the desired ligand (7), which is subsequently subjected to complexation with the suitable metal and optionally to salification: 
wherein R4 is as defined above for the compounds of formula (II).
2) Tetramerization of a suitably substituted N-benzylaziridine.
The general scheme for the synthesis of the ligand described in example 4 and of the derivatives thereof is reported below: 
wherein Bz=benzyl.
The cyclotetramerization of (R)-2-[(phenylmethoxy) methyl]-1-(phenylmethyl)aziridine (4) to give compound (5) is based on a photoinduced electron transfer mechanism by means of light emitted by a high-pressure mercury-vapor lamp, shielded from radiations of wavelength below 300 nm by a Pyrex filter; the reaction takes place in the presence of an oxidizing photochemical sensitizer, such as 9,10-dicyanoanthracene, and of catalytic amounts of an acid, such as 4-toluenesulfonic acid, at a temperature from room temperature to 60xc2x0 C. The reaction is preferably carried out in acetonitrile; alternatively, methanol or, preferably, an acetonitrile/methanol mixture, can be used.
The resulting compound (5) is debenzylated by catalytic hydrogenation in the subsequent step, to give intermediate (6), which is alkylated with the xcex1-halo derivative of a suitable carboxylic acid, to obtain the desired ligand (7), which is subsequently complexed with the suitable metal and, optionally, salified: 
wherein R4 is as defined above for compounds of formula (II).
Examples of cyclotetramerization of N-benzylaziridine exist in literature.
1. WO 95/31444, Example 7(C), discloses the synthesis of [2S-(2xcex1,5xcex1,8xcex1,11xcex1)]-2,5,8,11-tetramethyl-1,4,7,10-tetra-(phenylmethyl)-1,4,7,10-tetraazacyclododecane by cyclotetramerization of [S]-N-benzyl-2-methylaziridine with p-toluenesulfonic acid in ethanol as catalyst, at room temperature for 64-48 hours, followed by purification through column chromatography and alkalinization with NH4OH.
2. 1,4,7,10-Tetrabenzyl-1,4,7,10-tetraazacyclododecane was obtained by refluxing a mixture of N-benzylaziridine and p-toluenesulfonic acid in 95% ethanol for 6 hours (J. Heterocycl. Chem., 5(2), 305, 1968).
3. 1-Benzyl-2-(R)-ethylaziridine, by treatment with BF3Et2O for 20 hours at r.t., gave 1,4,7,10-tetrabenzyl-2,5,8,11-tetra-(R)-ethyl-1,4,7,10-tetraazacyclododecane; the same compound was obtained refluxing 1-benzyl-2-(R)-ethylaziridine in benzene or ethanol, for 24 hours, with the same catalyst (Tetrahedron Letters, 16, 1367-1370, 1970).
4. N-(Phenylethyl)aziridine, p-toluenesulfonic acid and aqueous ethanol heated to reflux for 25 hours gave 1,4,7,10-tetra(phenylethyl)-1,4,7,10-tetraazacyclododecane (U.S. Pat. No. 4,093,615).
On the other hand, the process for the cyclotetramerization of a suitably substituted N-benzylaziridine used for the preparation of [2S-(2R*,5R*,8R*,11R*)]-2,5,8,11-tetra [(phenylmethoxy)methyl]-1,4,7,10-tetra(phenylmethyl)-1,4,7,10-tetraazacyclododecane, an intermediate for the synthesis of the ligand described in example 4 of the present application, comprises a preliminary photochemical activation which unexpectedly provides a highly sterically hindered compound (Compound (5), Scheme 4). As far as the Applicant knows, this is the first example of cyclotetramerization of an N-benzyl-aziridine substituted with a hindered functionalized group (benzyloxymethyl) at one of the two ring carbon atoms.
Furthermore, and as much unexpectedly, all of the four asymmetric carbon atoms of the macrocyclic ring of the debenzylated compound (THM-Cyclen) have the same stereochemical configuration, as described in greater detail in the experimental section (Example 4E); therefore, the configuration of the stereogenic centre of the starting aziridine has been completely retained.
The gadolinium complexes the ligand described in example 4 (2,5,8,11-tetra(hydroxymethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (THM-DOTA)), in its four isomeric forms RRRR, RRRS, RRSS and RSRS, were the object of a recent article of molecular modelling, concerning theoretical calculations of molecular mechanics and molecular dynamic simulations (Eur. J. Med. Chem., 30, 539-546, 1995). This article deals with purely theoretical calculations: said compounds have never been synthesized actually. Thus, the compound is novel and its preparation is particularly original, both for its applicability and the possibility of obtaining compounds with controlled stereochemistry, although highly sterically hindered.
A non-limiting list of preferred ligands of the invention (which complexes with paramagnetic ions for use as M.R.I. contrast agents are described in the Experimental section) is reported in the following, to better exemplify the wide applicative potential of the present invention. 